Biallelic and gene-wide genomic substitution for endogenous intron and retroelement mutagenesis in human cells

Functional annotation of the vast noncoding landscape of the diploid human genome still remains a major challenge of genomic research. An efficient, scarless, biallelic, and gene-wide mutagenesis approach is needed for direct investigation of the functional significance of endogenous long introns in gene regulation. Here we establish a genome substitution platform, the Universal Knock-in System or UKiS, that meets these requirements. For proof of concept, we first used UKiS on the longest intron of TP53 in the pseudo-diploid cell line HCT116. Complete deletion of the intron, its substitution with mouse and zebrafish syntenic introns, and specific removal of retrotransposon-derived elements (retroelements) were all efficiently and accurately achieved in both alleles, revealing a suppressive role of intronic Alu elements in TP53 expression. We also used UKiS for TP53 intron deletion in human induced pluripotent stem cells without losing their stemness. Furthermore, UKiS enabled biallelic removal of all introns from three human gene loci of ~100 kb and longer to demonstrate that intron requirements for transcriptional activities vary among genes. UKiS is a standard platform with which to pursue the design of noncoding regions for genome writing in human cells.

Mismatched nucleotides and a missing nucleotide relative to the TP53-specific gRNA sequence are indicated in red. The PAM sequence is underlined.
b. The genomic locations of the off-target candidate sites. The primers used for genomic PCR are denoted by arrowheads. Locations are based on the human genome reference sequence hg38.
c. Representative images of genotyping PCR against the off-target sites by using genomic DNA from the indicated cell clones as the template. Genomic DNA was extracted from the positive clones obtained after the first step of UKiS. All clones were confirmed to have undergone successful recombination of the TP53 intron with the UKiS donors at both alleles (as shown in Fig. 2c). Source data are provided as a Source Data file. For the (a) intron-deleted clones, (b) mouse intron clones, and (c) zebrafish intron clones, graphical representation of the human TP53 locus is shown on the top: black boxes represent the positions of the homology arms used in our UKiS mutagenesis to TP53, horizontal lines flanked by two arrowheads represent the target regions for PCR, and the orange filled circles denote the positions of the heterozygous SNP site, rs12947788. Direct sequencing of the PCR genotyping amplicons indicated double peaks only at rs12947788 on the resultant sequencing chromatograms for all three clones for each kind of mutant.

UKiS (L)
UKiS 1st step product cell clone #1-10 UKiS (L)  Replacement of both UKiS donor alleles in clone #1-10 with one of two synthetic introns from which all retroelement (left) and Alu (right) sequences had been removed. First, the mutating payload plasmid containing the retroelement-or Alu-free intron and the TL-gRNA/Cas9 expression plasmid were transfected into clone #1-10. Thereafter, GFP-negative cells were collected by FACS and cloned. Biallelic substitution of UKiS markers with the mutating payload plasmid was confirmed by junction genotyping PCR that targeted the regions represented by horizontal lines flanked by two arrowheads in the schematic diagrams of the TP53 locus after successful replacement, with the expected length of PCR genotyping amplicons indicated.
In the agarose gel images, lane numbers of clones that underwent successful recombination in both alleles are in red. Source data are provided as a Source Data file.

PCR and direct sequencing
Retroelement-free intron cell clone # Real-time RT-PCR for transcriptional expression of five genes that are regulated by TP53 in the HCT116 cell clones having the TP53 first intron that are of full-length human, retroelement-free and Alu-free. Three different clones of each intron type that were tested in Fig. 6b were used here. As TP53-regulating genes, cyclin Replacement of both UKiS donor alleles with synthetic introns in which some of the Alu sequences or non-Alu sequences have been removed. First, the mutating payload plasmids containing the partial deletion of Alu sequences or deletion of non-Alu sequences from the first intron of TP53 and the TL-gRNA/Cas9 expression plasmid were transfected into clone #1-10. Thereafter, GFP-negative cells were collected by FACS and cloned. Biallelic substitution of UKiS markers with the mutating payload plasmid was confirmed by junction genotyping PCR, which targeted the regions represented by horizontal lines flanked by two arrowheads in the schematic diagrams of the TP53 locus after successful replacement.
In the agarose gel images, lane numbers of clones that underwent successful recombination in both alleles are in red.
Source data are provided as a Source Data file.

PCR and direct sequencing
Group III Alu deletion cell clone #

PCR and direct sequencing
Group II Alu deletion cell clone #  11b) and was used to create the deletion of the first intron of TP53 in iPS cell clones (Fig. 7a). Allele-specific PCR was performed by using primers for puromycin or blasticidin marker sequences, demonstrating that the puromycin and blasticidin alleles had A and G at rs1641548, respectively. Horizontal lines flanked by two arrowheads represent the target regions for the PCR of each allele. c. Schematic diagram of the second step of UKiS during which both UKiS donor alleles were replaced with the intron-free CD44 sequence. First, the intron-free CD44 payload plasmid and the TL-gRNA/Cas9 expression plasmid were transfected into clone #1-1, which was obtained in (b) and mostly GFP-positive (99.5%) as shown by FACS analysis. Thereafter, GFP-negative cells were collected by FACS and cloned. Biallelic substitution of UKiS markers with the mutating payload plasmid was confirmed by junction genotyping PCR ( Supplementary Fig. 16). A representative image of the resulting agarose gel electrophoresis of junction PCR amplicons is shown in Figure 7c. c. Schematic diagram of the second step of UKiS during which both UKiS donor alleles were replaced with the intron-free MET sequence. First, the intron-free MET payload plasmid and the TL-gRNA/Cas9 expression plasmid were transfected into clone #1-1, which was obtained in (b) and mostly GFP-positive (99.5%) as shown by FACS analysis. Thereafter, GFP-negative cells were collected by FACS and cloned. Biallelic substitution of UKiS markers with the mutating payload plasmid was confirmed by junction genotyping PCR ( Supplementary Fig. 17). A representative image of the resulting agarose gel electrophoresis of junction PCR amplicons is shown in Figure 7d.    Allele 2 left arm right arm rs142374380  TP53 UKiS 1st step product HCT116 cell clone #1-10 ( Fig. 3a) Human intron (Fig. 3b) 1st intron deleted (Fig. 3c) Mouse intron (Fig. 3d) Zebrafish intron (Fig. 3e) Retroelement-free ( Supplementary Fig. 6 TCACGTAAGTAGAACATGAAATAACC GAAGACGGCAGCAAAGAAAC Fig. 7c entire CD44 AGTGGATGGACAGGAGGATG GAGTGGGTCTGAGTGGGAAC Fig. 7d entire MET TGAAATCACTCTTATGTAACCTCTGG TGCAGGTATAGGCAGTGACAAG Fig. 7e entire  Supplementary Fig. 16 rs61882771 CCATTTTCAGTGGTCTGGATT Supplementary Fig. 17 rs6566 TCTGCTCTGTGGAAAGAAAGAA Supplementary Fig. 18 rs142374380 ATTTGGCAGCACTTCAGTTT